Insulin stimulates the rate of glucose transport into adipocytes 10 to 15-fold within ten minutes. This remarkable stimulation is due at least in part to the translocation of glucose transporters from within the cell to the plasma membrane. The long-term objective is to elucidate at the molecular level how insulin causes this translocation of transporters. The specific aims for the coming grant period are as follows. (1) We have recently developed an immunoadsorption method for the purification of vesicles that contain the insulin-responsive, intracellular glucose transporters. The cellular organelle that contains these transporters will be identified by biochemical and electron microscopic methods. The composition, arrangement, and function of the polypeptides in these vesicles will be determined. (2) The quantitative contribution that the translocation of transporters to the plasma membranes makes to the overall stimulation of cellular glucose transport by insulin will be assessed. (3) The question of whether the glucose transporter is rapidly recycling between the plasma membrane and the endosomal compartment will be answered. If recycling is occurring, the site of insulin action on the kinetics will be ascertained. To achieve aims (2) and (3), a method for labeling the extracelllular domain of the transporter with a membrane-impermeant reagent will be developed. (4) The nature of the signal between the insulin receptor and the transporter will be investigated. Possible roles for covalent modification of the transporter, or of a transporter-associated protein, and for an intracellular mediator will be examined. (5) A major effort will be made to develop a cell-free system in which the insulin- stimulated translocation of transporters occurs. if this succeeds, the components of the system will be purified. All these studies will be conducted with the 3T3-L1 adipocyte. The results of this project will contribute to our knowledge of the mechanism of insulin action. They will therefore be of relevance to diabetes, since this disease is due to insulin deficiency or insensitivity.